Osteogenic differentiation driven by osteoclasts and macrophages

Introduction Osteoclasts are bone-resorbing cells closely related to bone turnover, whereas different macrophage subtypes contribute to bone fracture healing. As osteoclasts and macrophages share the same hematopoietic origin, the difference between both cell types on osteoblast coupling, crosstalk extent and consequent bone formation remains poorly understood. This study compares the potential of primary cells that are routinely considered as osteoclast and macrophage cultures on their ability to support osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Methods Human Peripheral Blood Mononuclear Cells (hPBMCs) were used to obtain macrophage or osteoclast cultures using appropriate stimulatory factors. With different seeding densities of hPBMCs, conditioned media from macrophage or osteoclast cultures were harvested for comparative evaluation of effects thereof on the osteogenic differentiation of hMSCs. Specific cytological staining was used to qualitatively evaluate macrophage and osteoclast cultures. Additionally, quantitative data on hMSC proliferation, osteogenic differentiation and mineralization were obtained via biochemical assays. Results Conditioned medium from osteoclast cultures obtained via low hPBMCs seeding densities, but not from high hPBMCs seeding densities or macrophages, stimulated hMSC osteogenic differentiation and mineralization. Upon cellular crosstalk, both pre-differentiated osteoclasts and non-polarized macrophages equally supported early hMSC osteogenic differentiation and mineralization, as confirmed by increased alkaline phosphatase levels within 7 days and increased calcium content within 14 days in comparison with undifferentiated controls. Initial hPBMCs seeding density strongly influences osteoclastogenesis and the paracrine effect of the resultant osteoclast population on the osteogenic differentiation of hMSCs. In addition, only in indirect coculture, macrophages provide similar stimulatory effects as pre-differentiated osteoclasts on the osteogenic differentiation of MSCs and mineralization. Conclusion Our results demonstrate stimulatory effects of osteoclast conditioned medium on hMSC osteogenic differentiation, depending on initial hPBMC seeding density. In addition, we show that osteoclast and macrophage cultures contain pools of polarized macrophages, which may be involved in the osteogenic effects. Our data provide insight into bone tissue engineering approaches by using multicellular interactions related to bone remodeling and healing for the in vitro modulation of osteogenic differentiation